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Literature summary for 4.1.99.3 extracted from

  • Damiani, M.J.; Yalloway, G.N.; Lu, J.; McLeod, N.R.; ONeill, M.A.
    Kinetic stability of the flavin semiquinone in photolyase and cryptochrome-DASH (2009), Biochemistry, 48, 11399-11411.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
wild-type and mutants cloned into pET3a vectors and overexpressed in Escherichia coli BL21(DE3)pLysS cells Synechococcus elongatus PCC 7942 = FACHB-805

Protein Variants

Protein Variants Comment Organism
A385S is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Shows enhanced semiquinone stability Synechococcus elongatus PCC 7942 = FACHB-805
G389N is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 3 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH Synechococcus elongatus PCC 7942 = FACHB-805
M353Q is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Has little impact on semiquinone stability Synechococcus elongatus PCC 7942 = FACHB-805
W392Y is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 2 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH Synechococcus elongatus PCC 7942 = FACHB-805

Organism

Organism UniProt Comment Textmining
Synechococcus elongatus PCC 7942 = FACHB-805
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Purification (Commentary)

Purification (Comment) Organism
wild-type and mutants purified by centrifugation, sonication and gel filtration Synechococcus elongatus PCC 7942 = FACHB-805

Storage Stability

Storage Stability Organism
-80°C, 10 mM KH2PO4/K2HPO4 buffer, pH 7, 350 mM NaCl, 10% glycerol Synechococcus elongatus PCC 7942 = FACHB-805

Synonyms

Synonyms Comment Organism
cyclobutane pyrimidine dimer photolyase
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Synechococcus elongatus PCC 7942 = FACHB-805
photolyase
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Synechococcus elongatus PCC 7942 = FACHB-805

Cofactor

Cofactor Comment Organism Structure
FAD large kinetic isotope and pH effects on the rate constants for FAD semiquinone oxidation, which reveal that proton transfer is rate-limiting. Photolyase-specific residues, Trp392 and Gly389, independently ensure a high kinetic barrier to semiquinone reactivity in photolyase, possibly through interactions with the adenine moiety of FAD and/or adjusting the polarity of the binding site. These residues have a much greater impact on semiquinone reactivity than the more FAD proximal Met353 or Ser395 Synechococcus elongatus PCC 7942 = FACHB-805